| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online October 23, 2003; 10.1104/pp.103.020305 Plant Physiology 133:1198-1208 (2003) © 2003 American Society of Plant Biologists Engineering of a Water-Soluble Plant Cytochrome P450, CYP73A1, and NMR-Based Orientation of Natural and Alternate Substrates in the Active Site1Department of Plant Stress Response, Institute of Plant Molecular Biology, Centre National de la Recherche Scientifique-Unité Propre de Recherche 2357, Université Louis Pasteur, 28 rue Goethe, F–67000 Strasbourg, France (G.A.S., D.W.-R.); Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Université René Decartes, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8601, 45 rue des Saints-Pères, F–75270 Paris cedex 06, France (R.A., P.M.D.); and Institut Européen de Chimie et Biologie-Ecole polytechnique, 16, avenue Pey Berland, F–33607 Pessac cedex, France (M.B.)
CYP73A1 catalyzes cinnamic acid hydroxylation, a reaction essential for the synthesis of lignin monomers and most phenolic compounds in higher plants. The native CYP73A1, initially isolated from Jerusalem artichoke (Helianthus tuberosus), was engineered to simplify purification from recombinant yeast and improve solublity and stability in the absence of detergent by replacing the hydrophobic N terminus with the peptitergent amphipathic sequence PD1. Optimized expression and purification procedures yielded 4 mg engineered CYP73A1 L–1 yeast culture. This water-soluble enzyme was suitable for 1H-nuclear magnetic resonance (NMR) investigation of substrate positioning in the active site. The metabolism and interaction with the enzyme of cinnamate and four analogs were compared by UV-visible and 1H-NMR analysis. It was shown that trans-3-thienylacrylic acid, trans-2-thienylacrylic acid, and 4-vinylbenzoic acid are good ligands and substrates, whereas trans-4-fluorocinnamate is a competitive inhibitor. Paramagnetic relaxation effects of CYP73A1-Fe(III) on the 1H-NMR spectra of cinnamate and analogs indicate that their average initial orientation in the active site is parallel to the heme. Initial orientation and distances of ring protons to the iron do not explain the selective hydroxylation of cinnamate in the 4-position or the formation of single products from the thienyl compounds. Position adjustments are thus likely to occur during the later steps of the catalytic cycle.
Cytochromes P450 of the CYP73 family catalyze the 4-hydroxylation of cinnamic acid, an early and obligatory step in the biosynthesis of most phenolic compounds such as lignin monomers, flavonoids, coumarins, stilbenes, lignans, and tannins (Dixon, 2001  ). CYP73 proteins are present at high levels in all vascular plants, and up to 20% of the terrestrial plant biomass is processed through their active sites. The CYP73 family appeared early during plant evolution and does not display the multiple gene duplication and divergence that is evident for most other plant P450 families (Paquette et al., 2000). The importance of their physiological function thus seems to have exerted a constraint on CYP73 evolution and on the conservation of active site structure.
Very limited data are available on the structure of the active sites of plant P450s. Most of the extent information has been obtained using site-directed mutagenesis, guided by homology modeling on crystallized bacterial protein (Kahn et al., 2001
To date, only one membrane-bound P450 protein has been crystallized, and investigation of substrate docking in the active site of mammalian and other eukaryotic enzymes has relied on indirect methods such as homology models, site-directed mutagenesis, and adduct formation. We have built a model of CYP73A1, based on the structures of crystallized P450s (Schoch et al., 2003
Engineering of a Soluble and Stable Form of CYP73A1
Previous attempts at CYP73A1 purification (Gabriac et al., 1991
Analysis of total P450 content in recombinant yeast microsomes (Table I) shows that the N-terminal exchange for PD1 does neither significantly alter the membrane localization of CYP73A1 nor its level of expression in yeast. There is no PD1-P450 accumulation in the soluble fraction compared with the wild type. Introduction of the 4-His-tag however leads to a 20% to 40% decrease in protein expression.
Interaction spectra with cinnamic acid indicate that the structure of the substrate-binding site is essentially preserved in all constructs (Table I). The No conversion of P450 into P420 was detected with the constructs under standard conditions (i.e. in 10% [v/v] glycerol and at 20°C) for several hours. The stability of the PD1-CYP73A1–4His (PD73His) mutant was thus further investigated at low concentration of glycerol (3% [v/v]) and high concentration of dithionite (10 mg mL–1) at 30°C for 2 h. The half-life of the P450-CO complex in such drastic conditions was 95.1 ± 8 min for PD73His instead of 45 ± 7 min for CYP73A1. PD73His is expressed at high levels, has conserved substrate-binding properties, and shows an increased stability. Its behavior upon purification in the absence of detergent was thus investigated.
The levels of expression and stability of plant P450 enzymes in yeast were previously shown to be dependent on the co-expressed P450-reductase (Cabello-Hurtado et al., 1998
The highest specific expression and activity was obtained in WAT11, which also displays the highest cytochrome c reductase activity (Table II). As previously reported (Cabello-Hurtado et al., 1998
The purification protocol was derived from the method described by Schalk et al. (1999
Mass spectrometry analysis of the purified enzyme confirmed the absence of protein contaminants and detected no residual detergent. The determined mass of 59,263 Da was that expected for a protein devoid of posttranslational modification. Cinnamate saturation experiments performed with the purified enzyme led to the determination of a KD of 5 µM and to complete low- to high-spin conversion of the heme-iron ( It was possible to elute and concentrate PD73His in only 10% (v/v) glycerol and in the complete absence of detergent. Provided that the protein was kept at high ionic strength, and in contrast to the wild-type enzyme, no precipitation or enzyme loss by adsorption on the tube walls was observed upon storage. Exchange of the wild-type membrane anchor for PD1 thus results in a very significant increase in water solubility and stability of the purified protein.
The 1H-NMR spectrum of cinnamate is not well resolved. Thus the chemical displacement for the ring ortho-protons, and meta- and para-protons give a common signal. The plot of the observed relaxation rates as a function of the substrate-bound fraction for all the resonances of cinnamate (Fig. 3) did not allow independent determination of T1M for the aromatic protons. Thus the iron-proton distances calculated for H3/H4/H5 (Table III) are averaged for their presence probability, which cannot be estimated; the value found, 5.8 Å for these protons, put them closer to the heme than the ethylenic protons. However, H4, which is at the position of cinnamate attack, should be located closer to the iron than H3/H5. To test this hypothesis, substrate analogs with proton signals better differentiated at positions equivalent to C4, C3, and C5 of cinnamate were analyzed.
The four structural analogs of cinnamate likely to present better resolved 1H-NMR spectra were selected (Fig. 4), submitted to preliminary 1H-NMR analysis, and investigated for their binding properties to recombinant PD73His. The proton signals of all four molecules were distinct from those of glycerol. The thiophene ring protons of trans-2-thienylacrylic acid (2TA) and trans-3-thienylacrylic acid (3TA) gave independent signals. 4-Fluorocinnamate has no proton at position 4 and gives well separated signals for its ortho- and meta-protons. The 4-vinyl benzoate has two distinct vinylic protons.
Binding properties determined from UV-visible difference spectra of recombinant yeast microsomes (Table IV) show that the dissociation constants of the four enzyme-ligand complexes are very similar to those measured for cinnamate and that the values for maximal spectral changes determined with 3TA, 2TA, and 4-vinylbenzoic acid (4VB) are indicative of complete or almost complete (i.e. 87%–100%) low- to high-spin conversion of the enzyme. In agreement with these binding data, 3TA, 2TA, and 4VB were found to be both substrates of CYP73A1 and strong competitive inhibitors of its cinnamate 4-hydroxylase (C4H) activity (Table II; Fig. 5). However, neither 3TA nor 2TA, which are analogs of well-documented metabolic inactivators of mammalian drug-metabolizing enzymes (Minoletti et al., 1999
Titration of saturating amounts of the above substrates with increasing concentrations of soluble CYP73A1 allowed the calculation of T1M for each proton of these molecules. The T1M values and calculated iron-proton distances for the different CYP73A1 ligands are summarized in Table III. As predicted from the large iron spin transitions induced by the binding of the ligands (Table IV), they all bind close to the heme. The most consistent results were obtained with the strongest ligands of CYP73A1, CA, 4FC, 2TA, and 3TA. Information provided by all of the 1H-NMR analyses coincided to show that the differences in the distance to the iron of all protons of each ligand were small. This indicates that the ligands do not stand upright above the iron, but that their average position is roughly parallel to the heme (or that they move fast enough at a similar average distance). Protons located closest to the carboxylate suspected of anchoring the substrate on the protein are, as anticipated, the most distant to the iron. The expected positions of hydroxylation on each substrate are within 5.5 to 7.5 Å from the iron. Surprisingly, the aromatic or thiophene rings do not seem to be positioned so as to favor the oxidative attack at the 4 position of cinnamate or its equivalent in 2TA or 3TA. All ring protons appear nearly equidistant to the iron.
A three-dimensional model consisting of each substrate plus iron and incorporating the iron-proton distances in Table III was built. The starting conformations of the substrates used for constructing the model were drawn from x-ray crystallographic data or deduced from energy minimization calculations. Individual sets were then superimposed with constraints on the superimposition of the iron atoms, the carboxylate oxygens, and when possible, the geometrical centroid of the rings. In the best final conformation, obtained by performing superimposition based just on carboxylate and iron atoms, all of the potential hydroxylation sites were close, and carbon 4 of the cinnamic acid analogs was located from 5.5 to 7 Å from the iron (Fig. 6). The model was built assuming a stable orientation and position of the substrate in the active site. It must however be kept in mind that the substrate might be somehow mobile at this stage in its interaction with the protein and that the measured distances between the iron and the protons only reflect an average relative position. Incorporation of this predicted substrate orientation in a three-dimensional model of the CYP73A1 protein and directed mutagenesis confirmation of this orientation are described elsewhere (Schoch et al., 2003
High-yield purification of a plant P450 enzyme water soluble and stable enough for allowing NMR or crystallographic studies has not been reported. The first objective of this work was to modify the native form of CYP73A1 to engineer a protein with an intact catalytic center and substrate-binding properties that would be easy to purify and be stable in the absence of detergent. Exchange of the N-terminal membrane-anchoring segment of CYP73A1 for the amphipathic helix PD1 led to a highly expressed protein in yeast that remained bound to the endoplasmic reticulum membrane. Contrary to the previous report on the PD1-engineered CYP2A4 expressed in E. coli (Sueyoshi et al., 1995 ), there was no increase in protein expression nor obvious change in protein intracellular location, although the high expression of wild-type CYP73A1 in yeast microsomal fraction was conserved in the PD73His construct. The substrate-binding constants of the engineered protein and complete low- to high-spin conversion upon addition of substrate were also conserved. Structure and accessibility of the active site of the whole population of P450 protein was preserved. The partial activity loss could reflect an altered binding and orientation on the membrane, which results in nonoptimal contact with the P450 reductase and impaired electron transfer. A similar loss in activity was recorded for the modified CYP2A4 when reconstituted in an artificial liposome system. However, contrary to E. coli-expressed CYP2A4, active PD73His could not be solubilized with Na2CO3. Detergent was needed for initial protein solubilization, but could be completely omitted in latter stages of purification. The enzyme was stable upon storage in the absence of detergent and in glycerol concentrations as low as 10%.
PD1 was designed to form an amphipathic
Other strategies have been used to engineer soluble P450s expressed in E. coli, usually based on truncation of N-terminal transmembrane anchor before or after membrane insertion. Only one, so far, led to successful protein crystallization (Cosme and Johnson, 2000
Several aromatic protons of cinnamic acid, the natural substrate of CYP73A1, give equivalent signals on 1H-NMR spectra. Cinnamate analogs with substituted aromatic or heterocyclic rings were therefore tested as ligands and substrates of CYP73A1 to refine the NMR analysis and obtain more precise information on the positioning of the substrate in the active site. This led to the demonstration that 4-fluorocinnamate is a strong competitive inhibitor of the C4H, and that two thiophene propenoic acid analogs of cinnamate are good competitive inhibitors and substrates of the enzyme. Another analog, 4VB, behaved as a weaker ligand and poorer substrate of CYP73A1. All four ligands were included in the NMR analysis. The clearest information provided by this analysis is that the average initial orientation of the ligand in the active site of the resting Fe(III) protein is roughly parallel to the heme. The calculated distances of the different ring protons to the iron are very similar and in a 5.6 to 7.5 Å range. This is similar to the distances between hydroxylation sites and the heme iron reported for other P450 enzymes (Modi et al., 1995
It is possible that a bias leading to an overestimation of the distances was introduced if the exchange between the free and bound ligands is not fast enough to neglect the residence time for the protons near the paramagnetic site in the calculations. Most ring protons appear almost equidistant to the iron, whereas the enzyme exclusively attacks the 4-position of cinnamic acid. This rather suggests that further position adjustments occur during the next steps of the catalytic cycle. Evidence of such substrate movement has been reported upon interaction with the reductase (Modi et al., 1997
Chemicals CA, 2TA, 4VB, and NADPH were from Sigma (l'Isle d'Abeau Chesnes, France); trans-4-fluorocinnamate (4FC) was from Lancaster (Morecambe, UK); 3TA was from Maybridge (Cornwall, UK); and trans-[3-14C]cinnamate was from Isotopchim (Ganagobie, France).
The modified CYP73A1 cDNAs were generated by PCR, using as a template the double-stranded wild-type CYP73A1 coding sequence subcloned as an EcoRI-BamHI fragment into the pBluescript SK phagemid and the following primers: for exchanging the membrane-anchoring segment for a peptitergent sequence, sense (1) 5'-CAG GAT CCA TGG AAG AAT TAT TAA AAC AAG CTT TAC AAC AAG CTC AAC AAT TAT TAC AAC AAG CTC AAG AAT TAG CTA AAA AAA TAC TAA TCT CCA AAC TCC GCG G-3' and antisense (2) 5'-GGG AAT TCC CTT AAA ATG ACC TAG GTT TAG CTA CG-3'; for generation of a C-terminally 4-His-tagged protein, sense (3) 5'-GGG ATC CCA TGG ACC TCC TCC TCA TAG AAA AAA CCC TCG TCG-3' and antisense (4) 5'-ATC GGA ATT CCC TTA ATG ATG ATG ATG AAA TGA CCT AGG TTT AGC TAC G-3'. For generation of the double mutant with a peptitergent at the N terminus and 4-His at the C terminus, amplification was performed using the primers 1 and 4.
PCR mixtures (50 µL) contained 150 µM of each dNTP, 1 µM of each primer, 50 ng of template, 1.25 units of Pfu DNA polymerase (Stratagene, La Jolla, CA), 20 mM Tris-HCl, pH 8.75, 10 mM KCl, 100 mM (NH4)2SO4, 2 mM MgSO4, 0.1% (w/v) Triton X-100, and 0.1 g mL–1 bovine serum albumin. The polymerase was added after 5 min of preheating at 92°C. Thirty cycles of amplification (1 min at 92°C; 2 min at 52°C; and 2 min at 72°C) were completed by 10-min extension at 72°C. PCR provided full-length cDNAs with flanking EcoRI and BamHI sites that were purified on agarose gel, digested, and ligated into the shuttle vector pYeDP60 (Urban et al., 1990
Several modified strains of Brewer's yeast (Saccharomyces cerevisiae) were tested to optimize expression of the mutant proteins: W(R) (Truan et al., 1993
Yeast transformation and preparation of microsomes (high-density procedure) were described by Urban et al. (1994
P450 content was calculated from CO-reduced versus reduced difference spectra (Omura and Sato, 1964
CA hydroxylation was assayed using radiolabeled trans-[3-14C]cinnamic acid and thin-layer chromatography analysis of the metabolites (Reichhart et al., 1980 Metabolism of alternate subtrates was analyzed by HPLC. The assay contained, in a total volume of 200 µL, 100 mM sodium phosphate, pH 7.4, 80 µg of yeast microsomal protein, 150 to 400 µM of substrate, and 600 µM of NADPH. The reaction was incubated at 27°C for 20 to 60 min and was quenched by the addition of 20 µL of HCl 4 N. The products were extracted three times with 2 volumes of ether:petroleum ether (50:50, v/v), the organic phases were pooled and evaporated under argon, and the residue was dissolved in the initial mobile phase (acetonitrile:water:acetic acid (10:90:0.2, v/v). Reverse-phase HPLC analysis was performed on a LiChrosorb RP-18 column (Merck, 4 x 125 mm, 5 µm) at a flow rate of 1 mL min–1, with elution 5 min isocratic and then a 20-min linear gradient from 10% to 52% (v/v) acetonitrile. Microsomes of yeasts transformed with a void plasmid or incubations without NADPH were used as controls to check for the absence of CYP73-independent metabolism and to calculate yields of organic extraction. Wavelength of detection and retention times for substrate and product are, respectively: 2TA (300 nm), 10.8 and 14 min; 3TA (280 nm), 11.7 and 14 min; 4VB (268 nm), 12.1 and 15.7 min; and cinnamate (275 nm and radio-detection), 7.5 and 12 min.
Yeast microsomes resuspended in 20 mM Tris-HCl, pH 8, containing 30% (v/v) glycerol were solubilized by adding dropwise 1.25% (w/v) Emulgen 911 (Kao Atlas, Tokyo) and were stirred on ice for 15 min (Schalk et al., 1999 For purification of His-tagged CYP73A1 by affinity chromatography, 200 mg of Emulgen 911 solubilized protein (i.e. 40 nmol of P450) was diluted with 1 volume of 20 mM Tris-HCl, pH 8, containing 1 M NaCl and centrifuged at 100,000g for 45 min. The supernatant was applied at a flow rate of 0.1 mL min–1 to a 5-mL HiTrap-chelating column (Pharmacia, Uppsala) complexed with Ni2+ using the procedure recommended by the manufacturer and equilibrated in 20 mM Tris-HCl, pH 8, containing 0.5 M NaCl, 10 mM imidazole, 0.5% (w/v) Emulgen 911, and 10% (v/v) glycerol (buffer A). The column was then washed at a flow rate of 1 mL min–1, successively with 60 mL of buffer A, 40 mL of buffer A with 0.1% (w/v) Emulgen 911 and 20 mM imidazole, and 30 mL of 20 mM Tris-HCl, pH 8, 0.5 M NaCl, 10% (v/v) glycerol, and 60 mM imidazole. The protein was then eluted with 20 mL of 20 mM Tris-HCl, pH 8, containing 0.5 M NaCl, 10% (v/v) glycerol, and 50 mM His. For large-scale purification (starting from 150 nmol of P450), another protocol was developed, combining the Mono Q (5 mL) and chelating columns (1 mL), to avoid saturation of the Ni2+ column. Acidic proteins were bound to the MonoQ, but not the engineered CYP73A1 (pI calculated from the amino acid sequence = 9.78). Differences with the affinity chromatography protocol were in the dilution of the solubilized protein (5-fold) and in the first wash that was performed without NaCl. Before using NaCl buffer, the Mono Q column was discarded. NaCl was always needed to keep the protein in solution and to avoid precipitation in the purified fractions in low glycerol and in the absence of detergent.
SDS-PAGE analysis (Laemmli, 1970
Purified protein were quantified using the Pierce bicinchoninic acid assay. Heme content was calculated from the Soret band of the electronic absorption spectrum of the oxidized protein using the Mass spectroscopy analyses were performed (with 0.3 nmol of purified P450) by MALDI-TOF with a Reflex III (Bruker, Wissembourg, France) and by Electrospray Ionization, Time of flight with a LCT (Micromass) coupled to a HPLC apparatus SMART (Pharmacia).
Proton NMR measurements were made on a Bruker AMX250 spectrometer operating at 250.13 MHz, internally locked on the 2H signal of the solvent in a standard 5-mm tube; signals were again referenced to HDO at 4.75 ppm. Temperature was kept at 300° K. Substrates were solubilized at 40 mM in deuterated 100 mM sodium phosphate buffer containing 10% (v/v) glycerol and 0.5 M NaCl; pH was adjusted to 7.4 with NaOD. Cinnamate was completely soluble in these conditions and was assumed to be freely exchangeable at the sodium phosphate and NaCl concentrations used for the NMR relaxation experiments.
The 1H-NMR spectra (250 MHz, D2O,
Molecule protons bound near a paramagnetic center (here Fe3+) have their relaxation rates increased by the fluctuating magnetic moment of the electron spin. If ligands rapidly exchange with molecules in bulk solution, the observed longitudinal relaxation time will represent the weighted average of those of the protons from the molecules in bulk solution and bound to the enzyme. For saturating substrate concentrations:
The slope of a plot of T1obs–1 versus E0/(KD + S0) gives the value of T1b, which is correlated to the paramagnetic relaxation time (T1M) by the following equation
 m is the residence time of the substrate in the enzyme active site. Because m values greater than 10–4 s are unlikely for type I substrates with a dissociation constant of 5 to 100 µM, Equation 2 can be simplified into
The Solomon and Bloembergen (1956
 I and S are respectively the nuclear and electron giromagnetic ratio, h is the reduced Planck's constant, S is the total electron spin, c is the correlation time, which describes the process that modulates the electron-nuclear dipolar coupling, and  I and S are respectively the proton and the electron Larmor frequencies. The first term of this equation describes the dipolar interaction; the second one describes the contact interaction, which is always negligible because for the proton A/h < 1 MHz.
The correlation time
r is the rotational correlation time, s is the electron spin relaxation time, and m is the time for the chemical exchange. The fastest process in solution will contribute to c most significantly. In the case of hemoproteins with high-affinity substrates, we can assume that the substrate is in fast exchange (thus M << T1M); for an exchangeable proton, we can admit that S = c, and we have determined previously that it was approximately 2 x 10–10 s. Thus the term containing c becomes a constant, and the Solomon-Bloembergen can be rewritten in a simplified form as:
The proton longitudinal relaxation times (T1) were measured by standard inversion recovery sequence (180° -
T1 was calculated in the initial substrate solution, and in the presence of eight to 10 increasing amounts of purified P450 ranging from 0 to 6 µM. After the titration, the P450 was reduced with dithionite and complexed to CO. The conversion to its diamagnetic complex restored T1 values identical to T1 of P450-free substrate, indicating that no significant paramagnetic contribution due to impurities nor contribution of the diamagnetic part of the protein was involved. Total titration time was always less than 4 h. FeIICO versus FeII electronic spectra were also recorded at the end of the experiments to confirm protein integrity. No formation of P420 was detected, however formation of the CO complex was greatly slowed down compared with enzyme in absence of substrate. T1M were calculated as the slope of the curve T1obs–1 versus E0/(KD + nS0) for proton in the NMR spectrum. The proton-iron distances were then calculated as: r = (T1M*k)1/6 using k = 153 Å µs–1, a constant value that we had previously determined on the same instrument in the same experimental conditions (Poli-Scaife et al., 1997
Molecular modeling studies were carried out on a SGI Indy workstation. The geometries of the molecules were taken from the Cambridge Crystallographic Data bank (Allen and Motherwell, 2002
The skilled technical assistance of Monique Le Ret is greatly appreciated. Critical readings of the manuscript by Keith Griffin are gratefully acknowledged. The W(R) and WAT11 yeast strains and the pYeDP60 expression vector were kindly provided by Drs. Denis Pompon and Philippe Urban. Received January 10, 2003; returned for revision June 1, 2003; accepted August 13, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.020305.
1 This work was supported by the Centre National de la Recherche Scientifique Programme Chimie-Physique du Vivant and by a fellowship from the French Ministry of Research to G.A.S. * Corresponding author; e-mail daniele.werck{at}ibmp-ulp.ustrasbg.fr; fax 33–3–90–24–18–84.
Allen FH, Motherwell WDS (2002) Applications of the Cambridge Structural Database in organic chemistry and crystal chemistry. Acta Crystallogr Sect B 58: 407–422[Medline] Benveniste I, Gabriac B, Durst F (1986) Purification and characterization of the NADPH-cytochrome P-450 (cytochrome c) reductase from higher-plant microsomal fraction. Biochem J 235: 365–373[Medline]
Cabello-Hurtado F, Batard Y, Salaün JP, Durst F, Pinot F, Werck-Reichhart D (1998) Cloning, expression in yeast, and functional characterization of CYP81B1, a plant cytochrome P450 that catalyzes in-chain hydroxylation of fatty acids. J Biol Chem 273: 7260–7267
Cosme J, Johnson EF (2000) Engineering microsomal cytochrome P450 2C5 to be a soluble, monomeric enzyme: mutations that alter aggregation, phospholipid dependence of catalysis, and membrane binding. J Biol Chem 275: 2545–2553
Crull GB, Kennington JW, Garber AR, Ellis PD, Dawson JH (1989) F nuclear magnetic resonance as a probe of the spatial relationship between the heme iron of cytochrome P-450 and its substrate. J Biol Chem 264: 2649–2655 Dixon RA (2001) Natural products and plant disease resistance. Nature 411: 843–847[CrossRef][Medline] Duggleby RG (1984) Regression analysis of nonlinear Arrhenius plots: an empirical model and a computer program. Comput Biol Med 14: 447–455[CrossRef][Medline] Gabriac B, Werck-Reichhart D, Teutsch H, Durst F (1991) Purification and immunocharacterization of a plant cytochrome P450: the cinnamic acid 4-hydroxylase. Arch Biochem Biophys 288: 302–309[CrossRef][Medline] Kahn RA, Le Bouquin R, Pinot F, Benveniste I, Durst F (2001) A conservative amino acid substitution alters the regiospecificity of CYP94A2, a fatty acid hydroxylase from the plant Vicia sativa. Arch Biochem Biophys 391: 180–187[CrossRef][Web of Science][Medline] Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680–685[CrossRef][Medline]
Latunde-Dada AO, Cabello-Hurtado F, Czittrich N, Didierjean L, Schopfer C, Hertkorn N, Werck-Reichhart D, Ebel J (2001) Flavonoid 6-hydroxylase from soybean (Glycine max L.), a novel plant P-450 monooxygenase. J Biol Chem 276: 1688–1695 Li H, Poulos TL (1997) The structure of the cytochrome P450BM3 heam domain complexed with the fatty acid substrate, palmitoleic acid. Nat Struct Biol 4: 140–146[CrossRef][Web of Science][Medline] Mildvan AS, Gupta RK (1978) Binding free energy calculations for P450cam-substrate complexes. Methods Enzymol 49: 322–359[Medline] Minoletti C, Dijols S, Dansette PM, Mansuy D (1999) Comparison of the substrate specificities of human liver cytochrome P450s 2C9 and 2C18: application to the design of a specific substrate of CYP 2C18. Biochemistry 38: 7828–7836[CrossRef][Medline] Modi S, Gilham DE, Sutcliffe MJ, Lian LY, Primrose WU, Wolf CR, Roberts GC (1997) 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine as a substrate of cytochrome P450 2D6: allosteric effects of NADPH-cytochrome P450 reductase. Biochemistry 36: 4461–4470[CrossRef][Medline] Modi S, Paine MJ, Sutcliffe MJ, Lian LY, Primrose WU, Wolf CR, Roberts GC (1996a) A model for human cytochrome P450 2D6 based on homology modeling and NMR studies of substrate binding. Biochemistry 35: 4540–4550[CrossRef][Medline] Modi S, Primrose WU, Boyle JM, Gibson CF, Lian LY, Roberts GC (1995) NMR studies of substrate binding to cytochrome P450 BM3: comparisons to cytochrome P450 cam. Biochemistry 34: 8982–8988[CrossRef][Medline] Modi S, Sutcliffe MJ, Primrose WU, Lian LY, Roberts GC (1996b) The catalytic mechanism of cytochrome P450 BM3 involves a 6 A movement of the bound substrate on reduction. Nat Struct Biol 3: 414–417[CrossRef][Medline] Morrissey JH (1981) Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 117: 307–310[CrossRef][Web of Science][Medline]
Omura T, Sato R (1964) The carbon monoxide binding pigment of liver microsomes: I. Evidence for its hemoprotein nature. J Biol Chem 239: 2370–2378 Paquette SM, Bak S, Feyereisen R (2000) Intron-exon organization and phylogeny in a large superfamily, the paralogous cytochrome P450 genes of Arabidopsis thaliana. DNA Cell Biol 19: 307–317[CrossRef][Web of Science][Medline]
Paulsen MD, Ornstein RL (1993) Substrate mobility in thiocamphor-bound cytochrome P450cam: an explanation of the conflict between the observed product profile and the x-ray structure. Protein Eng 6: 359–365 Pierrel MA, Batard Y, Kazmaier M, Mignotte-Vieux C, Durst F, Werck-Reichhart D (1994) Catalytic properties of the plant cytochrome P450 CYP73 expressed in yeast: substrate specificity of a cinnamate hydroxylase. Eur J Biochem 224: 835–844[Medline] Poli-Scaife S, Attias R, Dansette PM, Mansuy D (1997) The substrate binding site of human liver cytochrome P450 2C9: an NMR study. Biochemistry 36: 12672–12682[CrossRef][Medline] Pompon D, Louerat B, Bronine A, Urban P (1996) Yeast expression of animal and plant P450s in optimized redox environments. Methods Enzymol 272: 51–64[CrossRef][Web of Science][Medline]
Reichhart D, Salaün JP, Benveniste I, Durst F (1980) Time course of induction of cytochrome P450, NADPH-cytochrome c reductase, and cinnamic acid hydroxylase by phenobarbital, ethanol, herbicides, and manganese in higher plant microsomes. Plant Physiol 66: 600–604 Regal KA, Nelson SD (2000) Orientation of caffeine within the active site of human cytochrome P450 1A2 based on NMR longitudinal (T1) relaxation measurements. Arch Biochem Biophys 384: 47–58[CrossRef][Medline]
Robineau T, Batard Y, Nedelkina S, Cabello-Hurtado F, LeRet M, Sorokine O, Didierjean L, Werck-Reichhart D (1998) The chemically inducible plant cytochrome P450 CYP76B1 actively metabolizes phenylureas and other xenobiotics Plant Physiol 118: 1049–1056 Sawada Y, Kinoshita K, Akashi T, Aoki T, Ayabe S (2002) Key amino acid residues required for aryl migration catalysed by the cytochrome P450 2-hydroxyisoflavanone synthase. Plant J 31: 555–564[CrossRef][Web of Science][Medline]
Schafmeister CE, Miercke LJ, Stroud RM (1993) Structure at 2.5 A of a designed peptide that maintains solubility of membrane proteins. Science 262: 734–738 Schalk M, Batard Y, Seyer A, Nedelkina S, Durst F, Werck-Reichhart D (1997) Design of fluorescent substrates and potent inhibitors of CYP73As, P450s that catalyze 4-hydroxylation of cinnamic acid in higher plants. Biochemistry 36: 15253–15261[CrossRef][Medline]
Schalk M, Cabello-Hurtado F, Pierrel MA, Atanossova R, Saindrenan P, Werck-Reichhart D (1998) Piperonylic acid, a selective, mechanism-based inactivator of the trans-cinnamate 4-hydroxylase: a new tool to control the flux of metabolites in the phenylpropanoid pathway. Plant Physiol 118: 209–218
Schalk M, Croteau R (2000) A single amino acid substitution (F363I) converts the regiochemistry of the spearmint (–)-limonene hydroxylase from a C6- to a C3-hydroxylase. Proc Natl Acad Sci USA 97: 11948–11953 Schalk M, Nedelkina M, Schoch G, Batard Y, Werck-Reichhart D (1999) Role of unusual amino acid residues in the proximal and distal heme regions of a plant P450, CYP73A1. Biochemistry 38: 6093–6103[Medline]
Schlichting I, Berendzen J, Chu K, Stock AM, Maves SA, Benson DE, Sweet RM, Ringe D, Petsko GA, Sligar SG (2000) The catalytic pathway of cytochrome P450cam at atomic resolution. Science 287: 1615–1622 Schoch GA, Attias R, Le Ret M, Werck-Reichhart D (2003) Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1: homology model guided site-directed mutagenesis. Eur J Biochem 270: 3684–3685[Medline] Solomon I, Bloembergen N (1956) Nuclear magnetic interactions in the HF molecule. J Chem Phys 25: 261–266[CrossRef] Sueyoshi T, Park LJ, Moore R, Juvonen RO, Negishi M (1995) Molecular engineering of microsomal P450 2a-4 to a stable, water-soluble enzyme. Arch Biochem Biophys 322: 265–271[CrossRef][Medline] Truan G, Cullin C, Reisdorf P, Urban P, Pompon D (1993) Enhanced in vivo monooxygenase activities of mammalian P450s in engineered yeast cells producing high levels of NADPH-P450 reductase and human cytochrome b5. Gene 125: 49–55[CrossRef][Web of Science][Medline] Urban P, Cullin C, Pompon D (1990) Maximizing the expression of mammalian cytochrome P-450 monooxygenase activities in yeast cells. Biochimie 72: 463–472[Medline] Urban P, Werck-Reichhart D, Teutsch HG, Durst F, Regnier S, Kazmaier M, Pompon D (1994) Characterization of recombinant plant cinnamate 4-hydroxylase produced in yeast: kinetic and spectral properties of the major plant P450 of the phenylpropanoid pathway. Eur J Biochem 222: 843–850[Web of Science][Medline] Werck-Reichhart D, Jones OTG, Durst F (1988) Haem synthesis during cytochrome P-450 induction in higher plants: 5-aminolaevulinic acid synthesis through a five-carbon pathway in Helianthus tuberosus tuber tissues aged in the dark. Biochem J 24: 473–480 Wester MR, Johnson EF, Marques-Soares C, Dijols S, Dansette PM, Mansuy D, Stout CD (2003) The structure of mammalian cytochrome P450 2C5 complexed with diclofenac at 2.1Å resolution: evidence for an induced fit model of substrate binding. Biochemistry 42: 9335–9345[CrossRef][Medline] Williams PA, Cosme J, Sridhar V, Johnson E, Mc Ree DE (2000) Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity. Mol Cell 5: 121–131[CrossRef][Web of Science][Medline]
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | PLANT PHYSIOLOGY® | THE PLANT CELL | |
|---|---|---|---|
TOP
ABSTRACT
RESULTS
A(peak-trough) were calculated from the saturation curves of difference spectra recorded upon cinnamate binding to the oxidized yeast microsomes. P450 and C4H activity were not detected in W(R) transformed with a void plasmid.
-helix with a flat hydrophobic surface (Schafmeister et al., 1993
[ppm]) are for VB: 7.88 (d, J2,3 = 8.2 Hz, 2H, H2), 7.59 (d, J2,3 = 8.2 Hz, 2H, H3), 6.88 (dd, Ja,b = 11.0 Hz, Ja,c = 17.8 Hz, 1H, Ha), 6.69 (d, Ja,c = 17.7 Hz, 1H, Hc), and 5.43 (d, Ja,b = 11.0 Hz, 1H, Hb); for 4FC: 7.66 (dd, J2,3 = 8.8 Hz, JH,F = 5.6 Hz, 2H, H3, and H5), 7.41 (d, J2',3' = 16.1 Hz, 1H, H3'), 7.21 (t, J2,3 = 8.7 Hz, 2H, H2, and H6), and 6.49 (d, J2',3' = 16.2 Hz, 2H, H2'); for 3TA: 7.67 (s, 1H, H2), 7.52 (d, J4,5 = 4.8 Hz, 1H, H5), 7.48 (d, J4,5 = 4.6 z, 1H, H4), 7.44 (d, J2',3' = 16.1 Hz, 1H, H3'), and 6.40 (d, J2',3' = 15.9 Hz, 1H, H2'); for 2TA: 7.56 (d, J2',3' = 15.5 Hz, 1H, H3'), 7.52 (d, J4,5 = 4.0 Hz, 1H, H5), 7.37 (d, J3,4 = 3.3 Hz, 1H, H3), 7.16 (t, J3,4,5 = 4.6 Hz, 1H, H4), and 6.35 (d, J2',3' = 15.8 Hz, 1H, H2'); CA is 7.66 (d, J2,3 = 5.2 Hz, 2H, C-2H, H6), 7.47–7.51 (m, 3H, H3, H4 andH5), 7.44 (d, J2',3' = 17.2 Hz, 1H, H3'), and 6.57 (d, J2',3' = 16.0 Hz, 1H, H2'). 
